Plant protein kinases

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a protein kinase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the protein kinase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the protein kinase in a transformed host cell.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/092,438, filed Jul. 10, 1998 now abandoned.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingprotein kinase enzymes in plants and seeds.

BACKGROUND OF THE INVENTION

Calcium can function as a signal which activates gene expression viastimulation of calcium-dependent protein kinases. Calcium-dependentprotein kinases (CDPKs) represent a family of protein kinases which areproposed to contain, in a single polypeptide, both a kinase domain andan adjoining calmodulin-like domain with four calcium-binding motifs(Harper, J. F., et al. (1991) Science 252:951-954). Some CDPK proteinskinase have been isolated that require calcium but not calmodulin foractivity. Research has shown that multiple CDPK isoforms are present inArabidopsis thaliana and other plants and that plant CDPKs may play apivotal role in the regulation of many cellular process such as stressresponse (WO 98/26045) and male gametophyte formation (WO 97/35968).

Glycogen synthase kinase-3 (GSK-3) in animal cells is a serine/threoninekinase protein, which is involved in the regulating the activity ofseveral transcription factors including the DNA-binding activity of thec-jun/AP1 transcription factor. AP1 is a transcription factor thatrecognizes a specific enhancer target DNA sequence and when bound toenhancer regions stimulates promoter transcriptional activity. AP1 iscomposed of several polypeptides of which c-jun is the major component.Several plant GSK-3 proteins have been identified that have homology toGSK-3 gene family of protein kinases (Bianchi et al. (1994) Mol. Gen.Genet. 242(3):337-345).

There is a great deal of interest in identifying the genes that encodeprotein kinase enzymes involved in the control of gene expression inplants. These genes may be used to modulate or control proteinexpression in plant cells. Accordingly, the availability of nucleic acidsequences encoding all or a portion of a regulatory protein kinase wouldfacilitate studies to better understand gene regulation in plants, andprovide genetic tools to permit more accurate control and manipulationof gene expression in plants.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding protein kinase enzymes. Specifically, this invention concernsan isolated nucleic acid fragment encoding a calcium dependentphosphorylase or glycogen synthase kinase-3 and an isolated nucleic acidfragment that is substantially similar to an isolated nucleic acidfragment encoding a calcium dependent phosphorylase or glycogen synthasekinase-3. In addition, this invention relates to a nucleic acid fragmentthat is complementary to the nucleic acid fragment encoding calciumdependent phosphorylase or glycogen synthase kinase-3.

An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a protein kinaseselected from the group consisting of calcium dependent phosphorylaseand glycogen synthase kinase-3.

In another embodiment, the instant invention relates to a chimeric geneencoding a calcium dependent phosphorylase or glycogen synthasekinase-3, or to a chimeric gene that comprises a nucleic acid fragmentthat is complementary to a nucleic acid fragment encoding a calciumdependent phosphorylase or glycogen synthase kinase-3, operably linkedto suitable regulatory sequences, wherein expression of the chimericgene results in production of levels of the encoded protein in atransformed host cell that is altered (i.e., increased or decreased)from the level produced in an untransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a calciumdependent phosphorylase or glycogen synthase kinase-3, operably linkedto suitable regulatory sequences. Expression of the chimeric generesults in production of altered levels of the encoded protein in thetransformed host cell. The transformed host cell can be of eukaryotic orprokaryotic origin, and include cells derived from higher plants andmicroorganisms. The invention also includes transformed plants thatarise from transformed host cells of higher plants, and seeds derivedfrom such transformed plants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of a calcium dependent phosphorylase orglycogen synthase kinase-3 in a transformed host cell comprising: a)transforming a host cell with a chimeric gene comprising a nucleic acidfragment encoding a calcium dependent phosphorylase or glycogen synthasekinase-3; and b) growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of altered levels of calciumdependent phosphorylase or glycogen synthase kinase-3 in the transformedhost cell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding a calcium dependent phosphorylase orglycogen synthase kinase-3.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 shows a comparison of the amino acid sequences set forth in SEQID NOs:2, 4, 6 and 8 and the Zea mays and Arabidopsis thaliana sequences(SEQ ID NOs:17 (gi 3320104), 18 (gi 1839597) and 19 (gi 3402722)).

FIG. 2 shows a comparison of the amino acid sequences set forth in SEQID NOs:10, 12, 14 and 16 the Arabidopsis thaliana and Medicago sativasequences (SEQ ID NOs:21 (gi 170711), 22 (gi 1709129), 23 (gi 1709127)and 24 (gi 1480078)).

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Protein Kinase Enzymes SEQ ID NO: Clone (Amino ProteinDesignation (Nucleotide) Acid) Calcium dependent cl15.pk0017.f7 1 2phosphorylase kinase Calcium dependent rlr24.pk0094.d10 3 4phosphorylase kinase Calcium dependent srm.pk0007.d4 5 6 phosphorylasekinase Calcium dependent wlm1.pk0020.e5 7 8 phosphorylase kinaseGlycogen Synthase Contig composed 9 10 kinase-3 of: csi1.pk0004.f7cta1n.pk0039.e3 p0003.cgpg179r p0005.cbmeh92rb p0005.cbmfa44rp0015.cdpes47r p0077.cpoae32r p0087.cppah52r p0102.cerbj25rp0127.cntcj58r p0128.cpibn73r Glycogen Synthase rls72.pk0014.a10 11 12kinase-3 Glycogen Synthase Contig composd 13 14 kinase-3 of:sdc1c.pk003.e7 sdp2c.pk018.c13 sdp2c.pk038.c4 sdp4c.pk021.j10ses2w.pk0013.b8 ses2w.pk0031.c6 sfl1.pk0046.c4 sfl1.pk130.p16sr1.pk0160.g1 Glycogen Synthase Contig composed 15 16 kinase-3 of:wdk1c.pk006.p1 wle1n.pk0007.f12 wle1n.pk0050.c9 wle1n.pk0106.g7wlk4.pk0007.c4 wlm0.pk0020.g7 wlmk1.pk0035.c6 wr1.pk0110.g6wr1.pk0149.c8 wre1n.pk0098.h11 wre1n.pk0115.h2

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “nucleic acid fragment” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. A nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLASTO/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a maimer different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several proteinkinase enzymes have been isolated and identified by comparison of randomplant cDNA sequences to public databases containing nucleotide andprotein sequences using the BLAST algorithms well known to those skilledin the art. The nucleic acid fragments of the instant invention may beused to isolate cDNAs and genes encoding homologous proteins from thesame or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other calcium dependent phosphorylase orglycogen synthase kinase-3 enzymes, either as cDNAs or genomic DNAs,could be isolated directly by using all or a portion of the instantnucleic acid fragments as DNA hybridization probes to screen librariesfrom any desired plant employing methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the instantnucleic acid sequences can be designed and synthesized by methods knownin the art (Maniatis). Moreover, the entire sequences can be useddirectly to synthesize DNA probes by methods known to the skilledartisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673; Loh et al. (1989) Science 243:217). Productsgenerated by the 3′ and 5′ RACE procedures can be combined to generatefull-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36: 1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of calcium dependent phosphorylase kinase andglycogen synthase kinase in those cells.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppresion technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded protein kinase enzyme. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 7).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4(1):37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:367 1), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 1 7:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library TissueClone cl15 Corn (Zea mays L.) 15 day old leaf blades c115.pk0017.f7 csilCorn (Zea mays L.) silk csi1.pk0004.f7 cta1n Corn (Zea mays L.) tassel*cta1n.pk0039.e3 p0003 Corn (Zea mays L.) premeiotic ear shoot,p0003.cgpg179r 0.2-4 cm p0005 Corn (Zea mays L.) immature earp0005.cbmeh92rb p0005.cbmfa44r p0015 Corn (Zea mays L.) 13 days afterp0015.cdpes47r pollenation embryo p0077 Corn (Zea mays L.) pollenp0077.cpoae32r p0087 Corn (Zea mays L.) 11 days after p0087.cppah52rpollenation pericarp* p0102 Corn (Zea mays L.) early meiosis tasselsp0102.cerbj25r 16-18 cm long* p0127 Corn (Zea mays L.) nucellus tissue,P0127.cntcj58r 5 days after silking* p0128 Corn (Zea mays L.) pooledprimary and p0128.cpibn73r secondary immature ear rlr24 Rice (Oryzasativa L.) leaf (15 days after rlr24.pk0094.d10 pollenation) 24 hoursafter infection of Magaporthe grisea strain 4360-R-62 (AVR2-YAMO);Resistant rls72 Rice (Oryza sativa L.) leaf (15 days afterr1572.pk0014.a10 germination) 72 hours after infection of Magaporthegrisea strain 4360-R-67 (avr2-yamo); Susceptible sdc1c Soybean (Glycinemax L.) developing sdc1c.pk0003.e7 cotyledon (3-5 mm) sdp2c Soybean(Glycine max L.) developing sdp2c.pk018.c13 pods 6-7 mm sdp2c.pk038.c4sdp4c Soybean (Glycine max L.) developing sdp4c.pk021.j10 pods 10-12 mmses2w Soybean (Glycine max L.) embryogenic ses2w.pk0013.b8 suspension 2weeks after subculture ses2w.pk0031.c6 sfl1 Soybean (Glycine max L.)immature sfl1.pk0046.c4 flower sfl1.pk130.p16 sr1 Soybean (Glycine maxL.) root library sr1.pk0160.g1 srm Soybean (Glycine max L.) rootmeristem srm.pk0007.d4 wdk1c Wheat (Triticum aestivum L.) developingwdk1c.pk006.p1 kemel, 3 days after anthesis wle1n Wheat (Triticumaestivum L.) leaf 7 day wle1n.pk0007.f12 old etiolated seedling*wle1n.pk0050.c9 wle1n.pk0106.g7 wlk4 Wheat (Triticum aestivum L.)seedlings wlk4.pk0007.c4 4 hr after treatment with fungicide** wlm0Wheat (Triticum aestivum L.) seedlings wlm0.pk0020.g7 0 hr afterinoculation with Erysiphe graminis f. sp tritici wlm1 Wheat (Triticumaestivum L.) seedlings wlm1.pk0020.e5 1 hr after inoculation withErysiphe graminis f. sp tritici wlmk1 Wheat (Triticum aestivum L.)seedlings wlmk1.pk0035.c6 1 hr after inoculation with Erysiphe graminisf. sp tritici and treatment with fungicide** wr1 Wheat (Triticumaestivum L.) root; wr1.pk0110.g6 7 day old seedling, light grownw1l.pk0149.c8 wre1n Wheat (Triticum aestivum L.) root; wre1n.pk0098.h117 day old seedling, light grown* wre1n.pk0115.h2 *These libraries werenormalized essentially as described in U.S. Pat. No. 5,482,845,incorporated herein by reference. **Fungicdie: application of6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods ofusing this compound are described in USSN 08/545,827, incorporatedherein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651). Theresulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescentsequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding protein kinase enzymes were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) NatureGenetics 3:266-272) provided by the NCBI. For convenience, the P-value(probability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Calcium DependentPhonphorvlase Kinase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to calciumdependent phosphorylase kinase from Zea mays (NCBI Identifier No. gi3320104 and gi 1839597) and Arabidopsis thaliana (NCBI Identifier No. gi3402722). Shown in Table 3 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or contigs assembled from two or moreESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toZea mays and Arabidopsis thaliana Calcium Dependent Phorphorylase KinaseClone Status BLAST pLog Score cl15.pk0017.f7 EST 50.50 (gi 3320104)rlr24.pk0094.d10 FIS >254.00 (gi 1839597) srm.pk0007.d4 EST 77.70 (gi3402722) wlm1.pk0020.e5 EST 55.70 (gi 3402722)

FIG. 1 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:2, 4, 6 and 8 and the Zea mays and Arabidopsis thalianasequences (SEQ ID NOs:17 (gi 3320104), 18 (gi 1839597) and 19 (gi3402722)). The data in Table 4 represents a calculation of the percentidentity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6 and8 and the Zea mays and Arabidopsis thaliana sequences (SEQ ID NOs:17, 18and 19).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toZea mays and Arabidopsis thaliana Calcium Dependent Phorphorylase KinaseSEQ ID NO. Percent Identity to 2 85% (gi 3320104) 4 87% (gi 1839597) 676% (gi 3402722) 8 87% (gi 3402722)

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASARGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a calcium dependent phosphorylasekinase. These sequences represent two new corn and rice sequencesencoding a calcium dependent phosphorylase kinase and the first soybeanand wheat sequences encoding a calcium dependent phosphorylase.

Example 4

Characterization of cDNA Clones Encoding Glycoaen Synthase Kinase TheBLASTX search using the EST sequences from clones listed in Table 5revealed similarity of the polypeptides encoded by the cDNAs to glycogensynthase kinase from Arabidopsis thaliana (NCBI Identifier No. gi 170711and gi 1480078) and Medicago sativa (NCBI Identifier No. gi 1709129 andgi 1709127). Shown in Table 5 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), or contigs assembled from two or moreESTs (“Contig”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toArabidopsis thaliana and Medicago sativa Glycogen Synthase Kinase CloneStatus BLAST pLog Score Contig composed of: Contig >254.00 (gi 1170711)csi1.pk0004.f7 cta1n.pk0039.e3 p0003.cgpg179r p0005.cbmch92rbp0005.cbmfa44r p0015.cdpes47r p0077.cpoae32r p0087.cppah52rp0102.cerbj25r p0127.cntcj58r p0128.cpibn73r rls72.pk0014.a10 EST 30.70(gi 1709129) Contig composd of: Contig >254.00 (gi 1709127)sdc1c.pk0003.e7 sdp2c.pk018.c13 sdp2c.pk038.c4 sdp4c.pk021.j10ses2w.pk0013.b8 ses2w.pk0031.c6 sfl1.pk0046.c4 sfl1.pk130.p16sr1.pk0160.g1 Contig composed of: Contig >254.00 (gi 1480078)wdk1c.pk006.p1 wle1n.pk0007.f12 wle1n.pk0050.c9 wle1n.pk0106.g7wlk4.pk0007.c4 wlm0.pk0020.g7 wlmk1.pk0035.c6 wr1.pk0110.g6wr1.pk0149.c8 wre1n.pk0098.h11 wre1n.k0115.h2

FIG. 2 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:10, 12, 14 and 16 the Arabidopsis thaliana and Medicagosaliva sequences (SEQ ID NOs:20 (gi 1170711), 21 (gi 1709129), 22 (gi1709127) and 23 (gi 1480078)). The data in Table 6 represents acalculation of the percent identity of the amino acid sequences setforth in SEQ ID NOs:10, 12 14 and 16 and the Arabidopsis thaliana andMedicago saliva sequences (SEQ ID NOs:20, 21, 22 and 23).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toArabidopsis thaliana and Medicago sativa Glycogen Synthase Kinase SEQ IDNO. Percent Identity to 10 84% (gi 1170711) 12 61% (gi 1709129) 14 86%(gi 1709127) 16 81% (gi 1480078)

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASARGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a glycogen synthase kinase. Thesesequences represent the first corn, rice, soybean and wheat sequencesencoding glycogen synthase kinase.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or Smal) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML 103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and Smal and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb Smal-Sall fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinotlricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™-PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the P subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J Biol. Chem. 261:9228-9238) can be used for expression ofthe instant polypeptides in transformed soybean. The phaseolin cassetteincludes about 500 nucleotides upstream (5′) from the translationinitiation codon and about 1650 nucleotides downstream (3′) from thetranslation stop codon of phaseolin. Between the 5′ and 3′ regions arethe unique restriction endonuclease sites Nco I (which includes the ATGtranslation initiation codon), Sma I, Kpn I and Xba I. The entirecassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 ,L CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 1 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the CDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 rum of approximately 1,IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

23 1 484 DNA Zea mays unsure (389) unsure (396) unsure (402) unsure(430) unsure (467) unsure (469) unsure (479) 1 gccagcccca gctccagccccaactcgtct gccgaggcgc tgcccaccag gccgcgtccc 60 aaggcgccgc cggtgaagcgcgtgtccagc gccgggctgc tggtcggctc ggtgctcaag 120 cgcaggacgg agaaccttaaggacaagtac agcctggggc ggcgcctcgg gcagggccag 180 ttcggcacca cgtacctgtgcgtggagcgg gccacgggca aggagttcgc gtgcaagtcc 240 atcctgaagc gcaactcgtcaccgacgacg acgtggagga cgtccgccgg gagatccaga 300 taatgcacca cctggcgggccaccccaacg tgatctccat ccgcggcgcc tacgaggacg 360 ccgtcgccgt gacctcgtcatggactctng gcggcngcga antgttcaag gatgtgcaga 420 agggcactan acgagagaaggccgcgagct cgcaggtatg tcgcgtntna ggcgtgcant 480 catg 484 2 101 PRT Zeamays UNSURE (62) 2 Pro Pro Val Lys Arg Val Ser Ser Ala Gly Leu Leu ValGly Ser Val 1 5 10 15 Leu Lys Arg Arg Thr Glu Asn Leu Lys Asp Lys TyrSer Leu Gly Arg 20 25 30 Arg Leu Gly Gln Gly Gln Phe Gly Thr Thr Tyr LeuCys Val Glu Arg 35 40 45 Ala Thr Gly Lys Glu Phe Ala Cys Lys Ser Ile LeuLys Xaa Leu Val 50 55 60 Thr Asp Asp Asp Val Glu Asp Val Arg Arg Glu IleGln Ile Met His 65 70 75 80 His Leu Ala Gly His Pro Asn Val Ile Ser IleArg Gly Ala Tyr Glu 85 90 95 Asp Ala Val Ala Val 100 3 2374 DNA Oryzasativa 3 gcacgagggc gtattccgat ttctctctct ctctcctctt cttcttcttcttcttcccca 60 acgagcgact cgcctccacc tcctcgacct ccacctcgcg aggcggcggtgcggggggcc 120 ccaaacccta accctaattc cgctgcgccc gcgcccgcgc ccgcgcgcgccgacaggctg 180 ttgttgttgt tgccatgggg cagtgctacg gcaagggcgc gtcggggaggacggcggacg 240 atgagggcgg ggtggtgacg gagcaccagt cgccgccgcc ggcgaacgggctgccgtcga 300 cgccgccgcg gcagcaggcg caggcgcagg cgcagcaggt ggggacgccgaggcggcgtg 360 ggagtaagtc cggatcgacg acgccggggc accagacgcc tggggtggcgtggccgagcc 420 cgtacccgtc cgggggcgcg agcccgctgc cggccggggt gtcgccgtcgccggcgaggt 480 cgacgcccag gaggttcttc aagcggccgt tcccgccgcc gtcgccggccaagcacataa 540 aggccacgct cgccaagagg ctgggtgggg ggaagcccaa ggaagggacgataccggagg 600 agggaggcgt gggcgctggc ggcggcggtg gaggggccgc ggatggggcggagacggaga 660 ggccattgga caagacgttc gggttctcga agaacttcgg cgcgaagtacgagctcggga 720 aggaggtggg gaggggccac ttcggacaca cttgctccgc cgtcgtcaagaagggcgagt 780 acaagggaca gaccgtcgcc gtcaagatca tcgccaaagc taagatgacaacggcaatat 840 ccattgagga tgttcgtaga gaagtaaaaa ttttgagagc gttatcagggcacaataatc 900 tcgtcaaatt ctatgatgca tgtgaggatg gcctcaatgt ctacattgtcatggaattat 960 gtgagggagg agaattgcta gacagaatat tagccagagg cgggagatacacagaggaag 1020 atgccaaagc gattgttgta cagattttga gcgtagtagc cttctgtcatcttcaggggg 1080 tagtgcatcg tgatttgaag ccagagaatt tccttttcac aaccagggatgaaaatgctc 1140 ccatgaagtt gattgatttt ggtctctctg atttcattag accagatgaaaggcttaatg 1200 atattgttgg aagtgcatat tatgttgccc cagaggtttt acacagatcatatagtatgg 1260 aagcagacat ttggagtata ggtgtcataa cgtacattct gctctgtggcagtcggccat 1320 tctgggcacg aacagaatca ggaatattcc gatctgtgtt gagagctgatcccaactttg 1380 atgattcacc gtggcctaca gtatcagctg aagctaagga ttttgtgaagagatttctga 1440 acaaagatta ccgcaaaaga atgaccgctg ttcaagcact gactcatccttggttgcgag 1500 atgaacaaag gcagatcccg ctggacatac tcatcttcag attaattaagcaatacctcc 1560 gcgctacacc tcttaaacgg ttggcattaa aggcactatc caaggctttaagggaagatg 1620 aacttttgta tctcaaactg cagtttaaac tgctcgaacc tagagatgggtttgtatcac 1680 ttgacaactt tcggacggca ctaacgcgat atttaactga tgctatgaaggaatcgaggg 1740 ttcttgaatt tttgcatgcg ttggaaccac ttgcatacag aagaatggactttgaagagt 1800 tctgtgccgc agcaatcagt ccttaccagc ttgaggcact ggaaaggtgggaggagattg 1860 ctggaacagc tttccagcaa tttgaacaag agggcaaccg agtcatatcagttgaggaat 1920 tagcacagga attaaatctt gctccaactc attactccat cgttcaagactggatcagaa 1980 aatccgatgg caagctaaac tttctcgggt ttaccaaatt tttacatggtgtcacaataa 2040 ggggctcaaa tacaagacgg cattaagcga tttgcaaaag aaaatgtattcttttctctt 2100 ctaattttaa agccgctcat tatgtgaccc tgattgatgt tttcccctcctgctcctatc 2160 cctctggtca atatgatcat tattcttgtt cgtgctgctg tcggctgttgtcatcatagt 2220 tttttgtaga gaatacatgt aaagatcttt tgtaatgaat cgaatgatatgtttgttcaa 2280 gaaatatagt gtcatgttgt tcttttttgc ccagtaaaaa aaaaaaaaaaaaaaatactc 2340 gaggcggggc cgtaccacat cccccccctc agcg 2374 4 623 PRTOryza sativa 4 Met Gly Gln Cys Tyr Gly Lys Gly Ala Ser Gly Arg Thr AlaAsp Asp 1 5 10 15 Glu Gly Gly Val Val Thr Glu His Gln Ser Pro Pro ProAla Asn Gly 20 25 30 Leu Pro Ser Thr Pro Pro Arg Gln Gln Ala Gln Ala GlnAla Gln Gln 35 40 45 Val Gly Thr Pro Arg Arg Arg Gly Ser Lys Ser Gly SerThr Thr Pro 50 55 60 Gly His Gln Thr Pro Gly Val Ala Trp Pro Ser Pro TyrPro Ser Gly 65 70 75 80 Gly Ala Ser Pro Leu Pro Ala Gly Val Ser Pro SerPro Ala Arg Ser 85 90 95 Thr Pro Arg Arg Phe Phe Lys Arg Pro Phe Pro ProPro Ser Pro Ala 100 105 110 Lys His Ile Lys Ala Thr Leu Ala Lys Arg LeuGly Gly Gly Lys Pro 115 120 125 Lys Glu Gly Thr Ile Pro Glu Glu Gly GlyVal Gly Ala Gly Gly Gly 130 135 140 Gly Gly Gly Ala Ala Asp Gly Ala GluThr Glu Arg Pro Leu Asp Lys 145 150 155 160 Thr Phe Gly Phe Ser Lys AsnPhe Gly Ala Lys Tyr Glu Leu Gly Lys 165 170 175 Glu Val Gly Arg Gly HisPhe Gly His Thr Cys Ser Ala Val Val Lys 180 185 190 Lys Gly Glu Tyr LysGly Gln Thr Val Ala Val Lys Ile Ile Ala Lys 195 200 205 Ala Lys Met ThrThr Ala Ile Ser Ile Glu Asp Val Arg Arg Glu Val 210 215 220 Lys Ile LeuArg Ala Leu Ser Gly His Asn Asn Leu Val Lys Phe Tyr 225 230 235 240 AspAla Cys Glu Asp Gly Leu Asn Val Tyr Ile Val Met Glu Leu Cys 245 250 255Glu Gly Gly Glu Leu Leu Asp Arg Ile Leu Ala Arg Gly Gly Arg Tyr 260 265270 Thr Glu Glu Asp Ala Lys Ala Ile Val Val Gln Ile Leu Ser Val Val 275280 285 Ala Phe Cys His Leu Gln Gly Val Val His Arg Asp Leu Lys Pro Glu290 295 300 Asn Phe Leu Phe Thr Thr Arg Asp Glu Asn Ala Pro Met Lys LeuIle 305 310 315 320 Asp Phe Gly Leu Ser Asp Phe Ile Arg Pro Asp Glu ArgLeu Asn Asp 325 330 335 Ile Val Gly Ser Ala Tyr Tyr Val Ala Pro Glu ValLeu His Arg Ser 340 345 350 Tyr Ser Met Glu Ala Asp Ile Trp Ser Ile GlyVal Ile Thr Tyr Ile 355 360 365 Leu Leu Cys Gly Ser Arg Pro Phe Trp AlaArg Thr Glu Ser Gly Ile 370 375 380 Phe Arg Ser Val Leu Arg Ala Asp ProAsn Phe Asp Asp Ser Pro Trp 385 390 395 400 Pro Thr Val Ser Ala Glu AlaLys Asp Phe Val Lys Arg Phe Leu Asn 405 410 415 Lys Asp Tyr Arg Lys ArgMet Thr Ala Val Gln Ala Leu Thr His Pro 420 425 430 Trp Leu Arg Asp GluGln Arg Gln Ile Pro Leu Asp Ile Leu Ile Phe 435 440 445 Arg Leu Ile LysGln Tyr Leu Arg Ala Thr Pro Leu Lys Arg Leu Ala 450 455 460 Leu Lys AlaLeu Ser Lys Ala Leu Arg Glu Asp Glu Leu Leu Tyr Leu 465 470 475 480 LysLeu Gln Phe Lys Leu Leu Glu Pro Arg Asp Gly Phe Val Ser Leu 485 490 495Asp Asn Phe Arg Thr Ala Leu Thr Arg Tyr Leu Thr Asp Ala Met Lys 500 505510 Glu Ser Arg Val Leu Glu Phe Leu His Ala Leu Glu Pro Leu Ala Tyr 515520 525 Arg Arg Met Asp Phe Glu Glu Phe Cys Ala Ala Ala Ile Ser Pro Tyr530 535 540 Gln Leu Glu Ala Leu Glu Arg Trp Glu Glu Ile Ala Gly Thr AlaPhe 545 550 555 560 Gln Gln Phe Glu Gln Glu Gly Asn Arg Val Ile Ser ValGlu Glu Leu 565 570 575 Ala Gln Glu Leu Asn Leu Ala Pro Thr His Tyr SerIle Val Gln Asp 580 585 590 Trp Ile Arg Lys Ser Asp Gly Lys Leu Asn PheLeu Gly Phe Thr Lys 595 600 605 Phe Leu His Gly Val Thr Ile Arg Gly SerAsn Thr Arg Arg His 610 615 620 5 568 DNA Glycine max unsure (11) unsure(69) unsure (83) unsure (95) unsure (148) unsure (196) unsure (272)unsure (557) unsure (563) unsure (568) 5 aaacccaagc ncccttcccagctggttcaa aaactcccct tcctcaaact caaaccctag 60 cagcgtcant caacacccttgcngatcttc aagcncccct tccctccgcc ctctccggcc 120 aagcacattc gcgcgctgctcgcccgcngc cacggttccg tcaagccgaa cgaagcctcc 180 ataccggagg ccagcnagtgtgagctcggc ctcgacaaga gctttggctt tgctaagcag 240 ttttcggctc attatgagctcagtgacgaa gngggccggg ggcattttgg gtatacctgc 300 tccgctaaag gcaagaaaggggcgttcaag ggcttaaatg ttgctgtcaa agtcattcct 360 aaagccaaga tgaccacagcaattgctata gaggatgtaa ggagagaagt gaagatattg 420 agggctttaa caggacataagaatctggtg caattctatg aagcctatga agatgatgac 480 atgtttatat agtttggagttgtgcaagga gggggaattg ctagatagga ttctttccgg 540 ggtggaaagt acctcgnagaggntgccn 568 6 157 PRT Glycine max UNSURE (4) UNSURE (22) UNSURE (25)UNSURE (29) UNSURE (47) UNSURE (63) UNSURE (88) 6 Asn Pro Ser Xaa LeuPro Ser Trp Phe Lys Asn Ser Pro Ser Ser Asn 1 5 10 15 Ser Asn Pro SerSer Xaa Pro Leu Xaa Ile Phe Lys Xaa Pro Phe Pro 20 25 30 Pro Pro Ser ProAla Lys His Ile Arg Ala Leu Leu Ala Arg Xaa His 35 40 45 Gly Ser Val LysPro Asn Glu Ala Ser Ile Pro Glu Ala Ser Xaa Cys 50 55 60 Glu Leu Gly LeuAsp Lys Ser Phe Gly Phe Ala Lys Gln Phe Ser Ala 65 70 75 80 His Tyr GluLeu Ser Asp Glu Xaa Gly Arg Gly His Phe Gly Tyr Thr 85 90 95 Cys Ser AlaLys Gly Lys Lys Gly Ala Phe Lys Gly Leu Asn Val Ala 100 105 110 Val LysVal Ile Pro Lys Ala Lys Met Thr Thr Ala Ile Ala Ile Glu 115 120 125 AspVal Arg Arg Glu Val Lys Ile Leu Arg Ala Leu Thr Gly His Lys 130 135 140Asn Leu Val Gln Phe Tyr Glu Ala Tyr Glu Asp Asp Asp 145 150 155 7 498DNA Triticum aestivum unsure (498) 7 cgaactactt gataagatat tggcgagaggtggaaagtat tctgaagagg atgcaaaggt 60 tgttatgctg caaattttga gtgtagtatcattttgccat cttcaaggtg ttgttcatcg 120 ggatctgaaa ccagagaatt ttctattctcatcgaaggag gaaaactcac ccttgaaggt 180 catagacttt ggcttgtctg actttgtaaagccagatgaa aggctcaacg acattgttgg 240 aagtgcgtat tatgttgctc cccgaggtgcttcatcgatc ttatggcacg gagggagata 300 tgttggagca ttggagtaat tgcctacattttgcttttgt gggaaccgac tttcctgggg 360 cacccacaga atccaggaat attcccagcttcccttaaag caaaaccaat tttgatgaag 420 ccccaaggcc tacctcctct gcggaaccaaagacttgtta aaagggtgct taataaggat 480 tacccaagag gatgacgn 498 8 111 PRTTriticum aestivum UNSURE (87) UNSURE (101) 8 Glu Leu Leu Asp Lys Ile LeuAla Arg Gly Gly Lys Tyr Ser Glu Glu 1 5 10 15 Asp Ala Lys Val Val MetLeu Gln Ile Leu Ser Val Val Ser Phe Cys 20 25 30 His Leu Gln Gly Val ValHis Arg Asp Leu Lys Pro Glu Asn Phe Leu 35 40 45 Phe Ser Ser Lys Glu GluAsn Ser Pro Leu Lys Val Ile Asp Phe Gly 50 55 60 Leu Ser Asp Phe Val LysPro Asp Glu Arg Leu Asn Asp Ile Val Gly 65 70 75 80 Ser Ala Tyr Tyr ValAla Xaa Glu Val Leu His Arg Ser Tyr Gly Thr 85 90 95 Glu Gly Asp Met XaaSer Ile Gly Val Ile Ala Tyr Ile Leu Leu 100 105 110 9 1814 DNA Zea mays9 gccatttccg gctttccgcc accacccccc ctctctctct ctctcttctt cttcttcaat 60cctccctccc cgcgccggag ttggaggagg gagaggggac aagctttccg gcgccgacgc 120cgacgcggac ccggcgccga cacgatccgg tggatcaagt gcatcacacc tttagggagg 180ccccttggac agcagtttgt gctgcaaatt ctatatagct ctgtcgcagc atggcctcgg 240tgggcgtggc acgctcttct ttgggatttc agaatggcac aagttctagc agtgacccag 300atcgtcttcc caacgagttg ggcagtatga gcataaggga cgacaaggac gttgaagata 360ttgtagtcaa tggcaatggg gcggagcctg gtcatatcat agtgaccagc attgatggga 420gaaatgggca ggcaaagcag accattagtt acatggctga gcgggtggta ggtcatgggt 480ccttcggaac cgttttccag gccaagtgtc ttgaaactgg tgagaccgta gctataaaaa 540aggttcttca agacaagaga tacaagaatc gtgagctgca aaccatgcga gtgcttgacc 600acccaaatgt ggtggctcta aagcactgtt tcttctcaaa gactgagaaa gaggagcttt 660acctcaattt ggtgcttgag tatgtaccgg agactgctca tcgtgtcatc aaacattaca 720acaagatgaa ccagcgcatg cctttgattt atgcaaaact gtatatgtat cagatttgta 780gagccttggc atacattcac aacagcattg gagtgtgcca cagggacatt aagccgcaaa 840atctcctggt taatcctcat acccatcagc taaaattgtg tgactttggc agcgcgaaag 900ttctggtaaa aggcgaacca aacatttctt acatctgttc taggtactac agagctccag 960agctcatatt tggtgctact gaatacacaa cagccattga tgttgggtct gctggctgtg 1020tgctcgctga gctgcttcta ggacagcctc tgttccctgg agaaagcggt gttgatcagc 1080ttgttgaaat catcaaggtt ctgggcacac ccacacgtga agaaattaag tgcatgaatc 1140caaattatac cgagtttaaa ttcccgcaaa tcaaagctca cccatggcat aagatattcc 1200ataaaaggat gcctgctgaa gcggtagatc tcgtgtccag gcttctgcag tactcaccaa 1260aacttcggtc gactgctttg gaagcattgg tccatccgtt ctttgatgaa cttcgggatc 1320caaacacccg cttaccgaat ggtcgttttc ttccgcctct cttcaatttt aagccccatg 1380agctgaagaa cgtgccggcg gatttcatgg tgaaattggt ccctgagcat gcacggaagc 1440aatgtgcctt cgtagggtgg tgatctctgg ataagaggat gacgactcga tgattagctg 1500aggaccaagt taatgtctgt tagaaactgc cggagatcga cattgccaga tgtggtgtgg 1560tataagatag gcaatatgtg tgattatttt ttgttcgagg ttatcacccc ccttgcccca 1620gaaaagatga gaagatgtcg atgtaacaag ccctctgcgc ttctgtaagt agatgagtgt 1680tgctgcatgc cccctgggta catgtatcgg tttgagcaga attctgtttg cctgaatcgt 1740gccatcacca cgcagggatc catcccttgt gtgacgatgt tcagcccaaa aaaaaaaaaa 1800aaaaaaaaaa aaaa 1814 10 410 PRT Zea mays 10 Met Ala Ser Val Gly Val AlaArg Ser Ser Leu Gly Phe Gln Asn Gly 1 5 10 15 Thr Ser Ser Ser Ser AspPro Asp Arg Leu Pro Asn Glu Leu Gly Ser 20 25 30 Met Ser Ile Arg Asp AspLys Asp Val Glu Asp Ile Val Val Asn Gly 35 40 45 Asn Gly Ala Glu Pro GlyHis Ile Ile Val Thr Ser Ile Asp Gly Arg 50 55 60 Asn Gly Gln Ala Lys GlnThr Ile Ser Tyr Met Ala Glu Arg Val Val 65 70 75 80 Gly His Gly Ser PheGly Thr Val Phe Gln Ala Lys Cys Leu Glu Thr 85 90 95 Gly Glu Thr Val AlaIle Lys Lys Val Leu Gln Asp Lys Arg Tyr Lys 100 105 110 Asn Arg Glu LeuGln Thr Met Arg Val Leu Asp His Pro Asn Val Val 115 120 125 Ala Leu LysHis Cys Phe Phe Ser Lys Thr Glu Lys Glu Glu Leu Tyr 130 135 140 Leu AsnLeu Val Leu Glu Tyr Val Pro Glu Thr Ala His Arg Val Ile 145 150 155 160Lys His Tyr Asn Lys Met Asn Gln Arg Met Pro Leu Ile Tyr Ala Lys 165 170175 Leu Tyr Met Tyr Gln Ile Cys Arg Ala Leu Ala Tyr Ile His Asn Ser 180185 190 Ile Gly Val Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val Asn195 200 205 Pro His Thr His Gln Leu Lys Leu Cys Asp Phe Gly Ser Ala LysVal 210 215 220 Leu Val Lys Gly Glu Pro Asn Ile Ser Tyr Ile Cys Ser ArgTyr Tyr 225 230 235 240 Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu TyrThr Thr Ala Ile 245 250 255 Asp Val Gly Ser Ala Gly Cys Val Leu Ala GluLeu Leu Leu Gly Gln 260 265 270 Pro Leu Phe Pro Gly Glu Ser Gly Val AspGln Leu Val Glu Ile Ile 275 280 285 Lys Val Leu Gly Thr Pro Thr Arg GluGlu Ile Lys Cys Met Asn Pro 290 295 300 Asn Tyr Thr Glu Phe Lys Phe ProGln Ile Lys Ala His Pro Trp His 305 310 315 320 Lys Ile Phe His Lys ArgMet Pro Ala Glu Ala Val Asp Leu Val Ser 325 330 335 Arg Leu Leu Gln TyrSer Pro Lys Leu Arg Ser Thr Ala Leu Glu Ala 340 345 350 Leu Val His ProPhe Phe Asp Glu Leu Arg Asp Pro Asn Thr Arg Leu 355 360 365 Pro Asn GlyArg Phe Leu Pro Pro Leu Phe Asn Phe Lys Pro His Glu 370 375 380 Leu LysAsn Val Pro Ala Asp Phe Met Val Lys Leu Val Pro Glu His 385 390 395 400Ala Arg Lys Gln Cys Ala Phe Val Gly Trp 405 410 11 584 DNA Oryza sativaunsure (4) unsure (206) unsure (240) unsure (308) unsure (328) unsure(333) unsure (344) unsure (372) unsure (377) unsure (387) unsure (396)unsure (417) unsure (498) unsure (506) unsure (521) 11 ggangaggccgcggctagcg agcgagcgag agagagggga gaagaagagg tgggacagcc 60 gggagatccatccctgtgga gaggagggag ggaggaagga ggcgttggag gaggagaggt 120 tgaccgatagatccattgcg gagttgagtg ttgatgcaaa gctgattcgc catcgtttag 180 ctttttataagagatgggtt cagtangggt tgcgccgtct gggttaaaca acagcagtan 240 caccagcatgggtgctgaga agttgcctga tcagatgcat gatctgaaga taagggacga 300 taaggaanttgaacgactat tattaacngc aanggaacag aaancggcca cataattgtc 360 acaactactggnggcanaaa tggtcanccg aaacanacag ttagctacat ggctgancgt 420 attgtagggcaaggttcatt tgggattgtc ttccaagcaa aattctggag acaaggtgag 480 acagttgctatcaagaangt tctcangata aacgctacaa naaccgttag cctcaaacca 540 tgcgccttcttgacaaccaa atgttgttac tcctgaagca tgtt 584 12 105 PRT Oryza sativa UNSURE(5) UNSURE (16) UNSURE (39) UNSURE (41)..(42) UNSURE (47)..(48) UNSURE(52) UNSURE (63) UNSURE (66) UNSURE (69) UNSURE (76) UNSURE (103) 12 MetGly Ser Val Xaa Val Ala Pro Ser Gly Leu Asn Asn Ser Ser Xaa 1 5 10 15Thr Ser Met Gly Ala Glu Lys Leu Pro Asp Gln Met His Asp Leu Lys 20 25 30Ile Arg Asp Asp Lys Glu Xaa Glu Xaa Xaa Thr Ile Ile Asn Xaa Xaa 35 40 45Gly Thr Glu Xaa Gly His Ile Ile Val Thr Thr Thr Gly Gly Xaa Asn 50 55 60Gly Xaa Pro Lys Xaa Thr Val Ser Tyr Met Ala Xaa Arg Ile Val Gly 65 70 7580 Gln Gly Ser Phe Gly Ile Val Phe Gln Ala Lys Phe Trp Arg Gln Gly 85 9095 Glu Thr Val Ala Ile Lys Xaa Val Leu 100 105 13 1429 DNA Glycine maxunsure (1202) unsure (1237) unsure (1297) unsure (1340) unsure (1376)unsure (1410) unsure (1416) 13 gcacaccaca caaaaaagca aaacagagagaacaactgtt actcacacac gccatgggta 60 aatgaatggt ttttgagcaa cagcagttaaaagagaaaag ggattcagcg aagatgacat 120 cggttggtgt ggcaccaact tcgggtttgagagaagccag tgggcatgga gcagcaggtg 180 ttgatagatt gccagaggag atgaacgatatgaaaattag ggatgataga gaaatggaag 240 ccacagttgt tgatggcaac ggaacggagacaggacatat cattgtgact accattgggg 300 gtagaaatgg tcagcccaag cagactataagctacatggc agagcgtgtt gtagggcatg 360 gatcatttgg agttgtcttc caggctaagtgcttggaaac cggtgaaact gtggctatca 420 aaaaggttct tcaagacaag aggtacaagaaccgggagct gcaaacaatg cgccttcttg 480 accacccaaa tgtcgttgct ttgaagcactgtttcttttc aaccactgaa aaggatgaac 540 tataccttaa tttggttctc gaatatgttcctgaaacagt taatcgggtg ataaaacatt 600 acaacaagtt taaccaaagg atgccactgatatatgtgaa actctataca taccagatct 660 ttagggcgtt atcttatatt catcgttgtattggagtctg ccatcgggat atcaagcctc 720 aaaatctatt ggtcaatcca cacactcaccaggttaaatt atgtgacttt ggaagtgcaa 780 aggttttggt aaaaggcgaa ccaaatatatcatacatatg ttctagatac tatagagcac 840 ctgagctcat atttggcgca actgaatatactacagccat tgacgtctgg tctgttggat 900 gtgttttagc tgagctgctg cttggacagcctctgttccc tggtgagagt ggagttgatc 960 aacttgttga gatcatcaag gttctgggcactccaacaag ggaagagatt aagtgcatga 1020 accctaatta tacagaattt aaattcccacagattaaagc acatccatgg cacaagatct 1080 tccataagcg catgcctcca gaggctgttgatttggtatc aagactacta caatactccc 1140 ctaacttgcg gtgcacagtt ttagatgccttggacgcacc ctttcctttg gacgaattcc 1200 gngatccaaa tcctcgcttg ccaaatgggccgatccntcc aacaactatt aattcaaacc 1260 catgaactga aagtgtccaa ctgagatttggggaaantgg tcaaagcatg caaggaacaa 1320 tgccgtttct ggcttgtaan tgtacaaaactgaagtgttg ttcatataga atgcgngctt 1380 cctcattaaa ggaattgtgg accttatgantcgttnccgt aacagttag 1429 14 399 PRT Glycine max UNSURE (391) 14 Met ValPhe Glu Gln Gln Gln Leu Lys Glu Lys Arg Asp Ser Ala Lys 1 5 10 15 MetThr Ser Val Gly Val Ala Pro Thr Ser Gly Leu Arg Glu Ala Ser 20 25 30 GlyHis Gly Ala Ala Gly Val Asp Arg Leu Pro Glu Glu Met Asn Asp 35 40 45 MetLys Ile Arg Asp Asp Arg Glu Met Glu Ala Thr Val Val Asp Gly 50 55 60 AsnGly Thr Glu Thr Gly His Ile Ile Val Thr Thr Ile Gly Gly Arg 65 70 75 80Asn Gly Gln Pro Lys Gln Thr Ile Ser Tyr Met Ala Glu Arg Val Val 85 90 95Gly His Gly Ser Phe Gly Val Val Phe Gln Ala Lys Cys Leu Glu Thr 100 105110 Gly Glu Thr Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Tyr Lys 115120 125 Asn Arg Glu Leu Gln Thr Met Arg Leu Leu Asp His Pro Asn Val Val130 135 140 Ala Leu Lys His Cys Phe Phe Ser Thr Thr Glu Lys Asp Glu LeuTyr 145 150 155 160 Leu Asn Leu Val Leu Glu Tyr Val Pro Glu Thr Val AsnArg Val Ile 165 170 175 Lys His Tyr Asn Lys Phe Asn Gln Arg Met Pro LeuIle Tyr Val Lys 180 185 190 Leu Tyr Thr Tyr Gln Ile Phe Arg Ala Leu SerTyr Ile His Arg Cys 195 200 205 Ile Gly Val Cys His Arg Asp Ile Lys ProGln Asn Leu Leu Val Asn 210 215 220 Pro His Thr His Gln Val Lys Leu CysAsp Phe Gly Ser Ala Lys Val 225 230 235 240 Leu Val Lys Gly Glu Pro AsnIle Ser Tyr Ile Cys Ser Arg Tyr Tyr 245 250 255 Arg Ala Pro Glu Leu IlePhe Gly Ala Thr Glu Tyr Thr Thr Ala Ile 260 265 270 Asp Val Trp Ser ValGly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln 275 280 285 Pro Leu Phe ProGly Glu Ser Gly Val Asp Gln Leu Val Glu Ile Ile 290 295 300 Lys Val LeuGly Thr Pro Thr Arg Glu Glu Ile Lys Cys Met Asn Pro 305 310 315 320 AsnTyr Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp His 325 330 335Lys Ile Phe His Lys Arg Met Pro Pro Glu Ala Val Asp Leu Val Ser 340 345350 Arg Leu Leu Gln Tyr Ser Pro Asn Leu Arg Cys Thr Val Leu Asp Ala 355360 365 Leu Asp Ala Pro Phe Pro Leu Asp Glu Phe Arg Asp Pro Asn Pro Arg370 375 380 Leu Pro Asn Gly Pro Ile Xaa Pro Thr Thr Ile Asn Ser Asn Pro385 390 395 15 1673 DNA Triticum aestivum unsure (1349) 15 aagtgtgagcccaccgtgtc cgccccattc acgccctagc cacatggagc atccggcgcc 60 ggcgccggagccgatgctgc tcgacgagca gccccccacc gcagtcgcct gcgagaagaa 120 gcagcaggatggcgaggcgc cgtatgcgga ggggaacgac gccatgaccg gtcacatcat 180 ctccaccaccatcggcggca agaacggcga gcccaagcag acgattagct acatggcgga 240 gcgcgttgtgggcactggtt cgtttggcat cgtctttcag gctaaatgcc tggaaaccgg 300 ggagatggtgggcattaaga aggtactgca ggacagacgg tacaagaacc gtgagctgca 360 gcttatgcgttcgatgatcc attccaatgt tgtctccctc aagcactgct tcttctcaac 420 cacaagtagagatgagctgt tcctgaacct tgtcatggag tatgtcccgg agacgctata 480 ccgcgtgcttaagcactaca gtaatgccaa ccaggggatg ccgcttatct atgtcaagct 540 ttacatgtatcagcttttta gagggctagc ttatgttcat actgttccag gagtttgcca 600 cagggatgtgaaaccacaaa atgttttggt tgatcctcta acccatcaag tcaagatctg 660 tgactttggaagtgcaaaag ttctggtacc tggtgaaccc aacatagcat acatatgctc 720 tcgctactatcgtgctcctg agctcatatt tggtgcaact gaatatacaa cttcaataga 780 catatggtcagctggatgtg ttcttgcaga gctacttctt ggtcagcctc tgtttccagg 840 agagactgcggttgatcagc tagtggagat tatcaaggtt cttggtactc caacccgtga 900 ggaaattcggtgcatgaacc ccaactatac cgagttcagg tttcctcaga ttaaggctca 960 tccttggcacaagattttcc acaagagaat gcccgctgaa gctatagatc ttgcctcccg 1020 ccttctccagtattcaccaa atctacgttg cactgctctt gatgcatgtg cacattcctt 1080 ctttgatgagctacgtgagc cgaatgcacg cttgccgaat ggccgcccat tccctcctct 1140 gttcaacttcaaacctgaac tagcgaacgc ctctccagag ctcatcaaca ggcttgttcc 1200 ggaacatgttcgacggcaaa atggccccaa cttcgcccat gctgggagct aaacggggcg 1260 cgcccgcatcgcccatattt ttgtttgtcc gccatcatcg aagaatcaat ctctccccta 1320 aatcctgaggagagaccgat caagtgcant gccagtgcca gtgaaagaag tacaactatg 1380 taaattacctgaccttggaa gaatcgttgt tgttgttgcc ggtgccggcc atgtttaagt 1440 acatggcggcacatgttggt tgagttgtta cttattatta agtaggtaag agcaatgatg 1500 taggaggtggagacatatgt taatgctagg tctgtgacct gttttaagta catttttgta 1560 atgcttggtagtggtactgt aatgcggcaa tagctgctcc atgttttgtc ccttgtccct 1620 gatgtaaatgtcgtcgtcct gcagcaaaaa aaaaaaaaaa aaaaaaaaaa aaa 1673 16 402 PRT Triticumaestivum 16 Met Glu His Pro Ala Pro Ala Pro Glu Pro Met Leu Leu Asp GluGln 1 5 10 15 Pro Pro Thr Ala Val Ala Cys Glu Lys Lys Gln Gln Asp GlyGlu Ala 20 25 30 Pro Tyr Ala Glu Gly Asn Asp Ala Met Thr Gly His Ile IleSer Thr 35 40 45 Thr Ile Gly Gly Lys Asn Gly Glu Pro Lys Gln Thr Ile SerTyr Met 50 55 60 Ala Glu Arg Val Val Gly Thr Gly Ser Phe Gly Ile Val PheGln Ala 65 70 75 80 Lys Cys Leu Glu Thr Gly Glu Met Val Gly Ile Lys LysVal Leu Gln 85 90 95 Asp Arg Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met ArgSer Met Ile 100 105 110 His Ser Asn Val Val Ser Leu Lys His Cys Phe PheSer Thr Thr Ser 115 120 125 Arg Asp Glu Leu Phe Leu Asn Leu Val Met GluTyr Val Pro Glu Thr 130 135 140 Leu Tyr Arg Val Leu Lys His Tyr Ser AsnAla Asn Gln Gly Met Pro 145 150 155 160 Leu Ile Tyr Val Lys Leu Tyr MetTyr Gln Leu Phe Arg Gly Leu Ala 165 170 175 Tyr Val His Thr Val Pro GlyVal Cys His Arg Asp Val Lys Pro Gln 180 185 190 Asn Val Leu Val Asp ProLeu Thr His Gln Val Lys Ile Cys Asp Phe 195 200 205 Gly Ser Ala Lys ValLeu Val Pro Gly Glu Pro Asn Ile Ala Tyr Ile 210 215 220 Cys Ser Arg TyrTyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu 225 230 235 240 Tyr ThrThr Ser Ile Asp Ile Trp Ser Ala Gly Cys Val Leu Ala Glu 245 250 255 LeuLeu Leu Gly Gln Pro Leu Phe Pro Gly Glu Thr Ala Val Asp Gln 260 265 270Leu Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Glu Ile 275 280285 Arg Cys Met Asn Pro Asn Tyr Thr Glu Phe Arg Phe Pro Gln Ile Lys 290295 300 Ala His Pro Trp His Lys Ile Phe His Lys Arg Met Pro Ala Glu Ala305 310 315 320 Ile Asp Leu Ala Ser Arg Leu Leu Gln Tyr Ser Pro Asn LeuArg Cys 325 330 335 Thr Ala Leu Asp Ala Cys Ala His Ser Phe Phe Asp GluLeu Arg Glu 340 345 350 Pro Asn Ala Arg Leu Pro Asn Gly Arg Pro Phe ProPro Leu Phe Asn 355 360 365 Phe Lys Pro Glu Leu Ala Asn Ala Ser Pro GluLeu Ile Asn Arg Leu 370 375 380 Val Pro Glu His Val Arg Arg Gln Asn GlyPro Asn Phe Ala His Ala 385 390 395 400 Gly Ser 17 639 PRT Zea mays 17Met Gly Asn Thr Cys Val Gly Pro Ser Ile Thr Met Asn Gly Phe Phe 1 5 1015 Gln Ser Val Ser Thr Ala Leu Trp Lys Thr Pro Gln Glu Gly Asp Ala 20 2530 Leu Pro Ala Ala Ala Asn Gly Pro Gly Gly Pro Ala Gly Ala Gly Ser 35 4045 Gln Ser Ala Leu Pro Lys Pro Ala Ser Asp Val His His Val Ala Val 50 5560 Gln Ser Glu Ala Pro Glu Pro Val Lys Ile Ala Ala Tyr His Ser Glu 65 7075 80 Pro Ala Pro Ala Val Arg Ser Glu Ala Pro Glu Pro Val Lys Ile Ala 8590 95 Ala Ser His Ser Glu Pro Ala Pro Met Ala Ala Lys Pro Gly Gly Ala100 105 110 Ala Ala Asn Ala Ser Pro Ser Pro Ser Pro Arg Pro Arg Pro GlnVal 115 120 125 Lys Arg Val Ser Ser Ala Gly Leu Leu Leu Gly Ser Val LeuArg Arg 130 135 140 Lys Thr Glu Asn Leu Lys Asp Lys Tyr Ser Leu Gly ArgArg Leu Gly 145 150 155 160 Gln Gly Gln Phe Gly Thr Thr His Leu Cys ValGlu Arg Ala Thr Gly 165 170 175 Lys Glu Leu Ala Cys Lys Ser Ile Leu LysArg Lys Leu Gly Ser Asp 180 185 190 Asp Asp Val Glu Asp Val Arg Arg GluIle Gln Ile Met His His Leu 195 200 205 Ala Gly His Pro Ser Val Val GlyIle Arg Gly Ala Tyr Glu Asp Ala 210 215 220 Val Ala Val His Leu Val MetGlu Leu Cys Gly Gly Gly Glu Leu Phe 225 230 235 240 Asp Arg Ile Val ArgArg Gly His Tyr Thr Glu Arg Lys Ala Ala Glu 245 250 255 Leu Ala Arg ValIle Val Gly Val Val Glu Ala Cys His Ser Met Gly 260 265 270 Val Met HisArg Asp Leu Lys Pro Glu Asn Phe Leu Phe Ala Asp His 275 280 285 Ser GluGlu Ala Ala Leu Lys Thr Ile Asp Phe Gly Leu Ser Ile Phe 290 295 300 PheArg Pro Gly Gln Ile Phe Thr Asp Val Val Gly Ser Pro Tyr Tyr 305 310 315320 Val Ala Pro Glu Val Leu Lys Lys Arg Tyr Gly Pro Glu Ala Asp Val 325330 335 Trp Ser Ala Gly Val Ile Ile Tyr Ile Leu Leu Cys Gly Val Pro Pro340 345 350 Phe Trp Ala Glu Asn Glu Gln Gly Ile Phe Glu Glu Val Leu HisGly 355 360 365 Arg Leu Asp Phe Glu Ser Glu Pro Trp Pro Ser Ile Ser AspGly Ala 370 375 380 Lys Asp Leu Val Arg Arg Met Leu Val Arg Asp Pro ArgLys Arg Leu 385 390 395 400 Thr Ala His Glu Val Leu Arg His Pro Trp ValGln Val Gly Gly Val 405 410 415 Ala Pro Asp Arg Pro Leu Asp Ser Ala ValLeu Ser Arg Met Lys Gln 420 425 430 Phe Ser Ala Met Asn Lys Leu Lys LysMet Ala Leu Arg Val Ile Ala 435 440 445 Glu Asn Leu Ser Glu Asp Glu IleAla Gly Leu Arg Glu Met Phe Lys 450 455 460 Met Ile Asp Ala Asp Asn SerGly Gln Ile Thr Phe Glu Glu Leu Lys 465 470 475 480 Val Gly Leu Glu LysVal Gly Ala Asn Leu Gln Glu Ser Glu Ile Tyr 485 490 495 Ala Leu Met GlnAla Ala Asp Val Asp Asn Asn Gly Thr Ile Asp Tyr 500 505 510 Gly Glu PheIle Ala Ala Thr Leu His Leu Asn Lys Val Glu Arg Glu 515 520 525 Asp HisLeu Phe Ala Ala Phe Gln Tyr Phe Asp Lys Asp Gly Ser Gly 530 535 540 TyrIle Thr Ala Asp Glu Leu Gln Val Ala Cys Glu Glu Phe Gly Leu 545 550 555560 Gly Asp Val Gln Leu Glu Asp Leu Ile Gly Glu Val Asp Gln Asp Asn 565570 575 Asp Gly Arg Ile Asp Tyr Asn Glu Phe Val Ala Met Met Gln Lys Pro580 585 590 Thr Val Gly Gly Ser Arg Arg Arg Pro Ile Cys Arg Thr Ala SerAla 595 600 605 Ser Gly Ser Ala Ser Gly Ser Gly Arg Arg Ser Gly Trp ProArg Pro 610 615 620 Leu Cys Leu Trp Leu Pro Cys Cys Leu Arg Val Gly ValAsp Asp 625 630 635 18 625 PRT Zea mays 18 Met Gly Gln Cys Tyr Gly LysAla Arg Gly Ala Ser Ser Arg Ala Asp 1 5 10 15 His Asp Ala Asp Pro SerGly Ala Gly Ser Val Ala Pro Pro Ser Pro 20 25 30 Leu Pro Ala Asn Gly AlaPro Leu Pro Ala Thr Pro Arg Arg His Lys 35 40 45 Ser Gly Ser Thr Thr ProVal His His His Gln Ala Ala Thr Pro Gly 50 55 60 Ala Ala Ala Trp Pro SerPro Tyr Pro Ala Gly Gly Ala Ser Pro Leu 65 70 75 80 Pro Ala Gly Val SerPro Ser Pro Ala Arg Ser Thr Pro Arg Arg Phe 85 90 95 Phe Lys Arg Pro PhePro Pro Pro Ser Pro Ala Lys His Ile Lys Ala 100 105 110 Thr Leu Ala LysArg Leu Gly Gly Gly Lys Pro Lys Glu Gly Thr Ile 115 120 125 Pro Glu GluGly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 130 135 140 Gly AlaAla Val Gly Ala Ala Asp Ser Ala Glu Ala Asp Arg Pro Leu 145 150 155 160Asp Lys Thr Phe Gly Phe Ala Lys Asn Phe Gly Ala Lys Tyr Asp Leu 165 170175 Gly Lys Glu Val Gly Arg Gly His Phe Gly His Thr Cys Ser Ala Val 180185 190 Val Lys Lys Gly Glu His Lys Gly His Thr Val Ala Val Lys Ile Ile195 200 205 Ser Lys Ala Lys Met Thr Thr Ala Ile Ser Ile Glu Asp Val ArgArg 210 215 220 Glu Val Lys Ile Leu Lys Ala Leu Ser Gly His Asp Asn LeuVal Arg 225 230 235 240 Phe Tyr Asp Ala Cys Glu Asp Ala Leu Asn Val TyrIle Val Met Glu 245 250 255 Leu Cys Glu Gly Gly Glu Leu Leu Asp Arg IleLeu Ala Arg Gly Gly 260 265 270 Arg Tyr Thr Glu Glu Asp Ala Lys Ala IleIle Val Gln Ile Leu Ser 275 280 285 Val Val Ala Phe Cys His Leu Gln GlyVal Val His Arg Asp Leu Lys 290 295 300 Pro Glu Asn Phe Leu Phe Thr ThrArg Asp Glu Ser Ala Pro Met Lys 305 310 315 320 Leu Ile Asp Phe Gly LeuSer Asp Phe Ile Arg Pro Asp Glu Arg Leu 325 330 335 Asn Asp Ile Val GlySer Ala Tyr Tyr Val Ala Pro Glu Val Leu His 340 345 350 Arg Ser Tyr SerMet Glu Ala Asp Ile Trp Ser Ile Gly Val Ile Thr 355 360 365 Tyr Ile LeuLeu Cys Gly Ser Arg Pro Phe Trp Ala Arg Thr Glu Ser 370 375 380 Gly IlePhe Arg Ser Val Leu Arg Ala Asp Pro Asn Phe Asp Asp Ser 385 390 395 400Pro Trp Pro Ser Val Ser Ala Glu Ala Lys Asp Phe Val Lys Arg Phe 405 410415 Leu Asn Lys Asp Tyr Arg Lys Arg Met Thr Ala Val Gln Ala Leu Thr 420425 430 His Pro Trp Leu Arg Asp Glu Gln Arg Gln Ile Pro Leu Asp Ile Leu435 440 445 Ile Phe Arg Leu Val Lys Gln Tyr Leu Arg Ala Thr Pro Leu LysArg 450 455 460 Leu Ala Leu Lys Ala Leu Ser Lys Ala Leu Ser Glu Asp GluLeu Leu 465 470 475 480 Tyr Leu Arg Leu Gln Phe Lys Leu Leu Glu Pro ArgAsp Gly Phe Val 485 490 495 Ser Leu Asp Asn Phe Arg Thr Ala Leu Thr ArgTyr Ser Thr Asp Ala 500 505 510 Met Arg Glu Ser Arg Val Leu Glu Phe GlnHis Ala Leu Glu Pro Leu 515 520 525 Ala Tyr Arg Lys Met Asp Phe Glu GluPhe Cys Ala Ala Ala Ile Ser 530 535 540 Pro Tyr Gln Leu Glu Ala Leu GluArg Trp Glu Glu Ile Ala Gly Thr 545 550 555 560 Ala Phe Gln His Phe GluGln Glu Gly Asn Arg Val Ile Ser Val Glu 565 570 575 Glu Leu Ala Gln GluLeu Asn Leu Ala Pro Thr His Tyr Ser Ile Val 580 585 590 Gln Asp Trp IleArg Lys Ser Asp Gly Lys Leu Asn Phe Leu Gly Phe 595 600 605 Thr Lys PheLeu His Gly Val Thr Ile Arg Gly Ser Asn Thr Arg Arg 610 615 620 His 62519 576 PRT Arabidopsis thaliana 19 Met Gly Ile Cys His Gly Lys Pro ValGlu Gln Gln Ser Lys Ser Leu 1 5 10 15 Pro Val Ser Gly Glu Thr Asn GluAla Pro Thr Asn Ser Gln Pro Pro 20 25 30 Ala Lys Ser Ser Gly Phe Pro PheTyr Ser Pro Ser Pro Val Pro Ser 35 40 45 Leu Phe Lys Ser Ser Pro Ser ValSer Ser Ser Val Ser Ser Thr Pro 50 55 60 Leu Arg Ile Phe Lys Arg Pro PhePro Pro Pro Ser Pro Ala Lys His 65 70 75 80 Ile Arg Ala Phe Leu Ala ArgArg Tyr Gly Ser Val Lys Pro Asn Glu 85 90 95 Val Ser Ile Pro Glu Gly LysGlu Cys Glu Ile Gly Leu Asp Lys Ser 100 105 110 Phe Gly Phe Ser Lys GlnPhe Ala Ser His Tyr Glu Ile Asp Gly Glu 115 120 125 Val Gly Arg Gly HisPhe Gly Tyr Thr Cys Ser Ala Lys Gly Lys Lys 130 135 140 Gly Ser Leu LysGly Gln Glu Val Ala Val Lys Val Ile Pro Lys Ser 145 150 155 160 Lys MetThr Thr Ala Ile Ala Ile Glu Asp Val Ser Arg Glu Val Lys 165 170 175 MetLeu Arg Ala Leu Thr Gly His Lys Asn Leu Val Gln Phe Tyr Asp 180 185 190Ala Phe Glu Asp Asp Glu Asn Val Tyr Ile Val Met Glu Leu Cys Lys 195 200205 Gly Gly Glu Leu Leu Asp Lys Ile Leu Gln Arg Gly Gly Lys Tyr Ser 210215 220 Glu Asp Asp Ala Lys Lys Val Met Val Gln Ile Leu Ser Val Val Ala225 230 235 240 Tyr Cys His Leu Gln Gly Val Val His Arg Asp Leu Lys ProGlu Asn 245 250 255 Phe Leu Phe Ser Thr Lys Asp Glu Thr Ser Pro Leu LysAla Ile Asp 260 265 270 Phe Gly Leu Ser Asp Tyr Val Lys Pro Asp Glu ArgLeu Asn Asp Ile 275 280 285 Val Gly Ser Ala Tyr Tyr Val Ala Pro Glu ValLeu His Arg Thr Tyr 290 295 300 Gly Thr Glu Ala Asp Met Trp Ser Ile GlyVal Ile Ala Tyr Ile Leu 305 310 315 320 Leu Cys Gly Ser Arg Pro Phe TrpAla Arg Thr Glu Ser Gly Ile Phe 325 330 335 Arg Ala Val Leu Lys Ala GluPro Asn Phe Glu Glu Ala Pro Trp Pro 340 345 350 Ser Leu Ser Pro Glu AlaVal Asp Phe Val Lys Arg Leu Leu Asn Lys 355 360 365 Asp Tyr Arg Lys ArgLeu Thr Ala Ala Gln Ala Leu Cys His Pro Trp 370 375 380 Leu Val Gly SerHis Glu Leu Lys Ile Pro Ser Asp Met Ile Ile Tyr 385 390 395 400 Lys LeuVal Lys Val Tyr Ile Met Ser Thr Ser Leu Arg Lys Ser Ala 405 410 415 LeuAla Ala Leu Ala Lys Thr Leu Thr Val Pro Gln Leu Ala Tyr Leu 420 425 430Arg Glu Gln Phe Thr Leu Leu Gly Pro Ser Lys Asn Gly Tyr Ile Ser 435 440445 Met Gln Asn Tyr Lys Thr Ala Ile Leu Lys Ser Ser Thr Asp Ala Met 450455 460 Lys Asp Ser Arg Val Phe Asp Phe Val His Met Ile Ser Cys Leu Gln465 470 475 480 Tyr Lys Lys Leu Asp Phe Glu Glu Phe Cys Ala Ser Ala LeuSer Val 485 490 495 Tyr Gln Leu Glu Ala Met Glu Thr Trp Glu Gln His AlaArg Arg Ala 500 505 510 Tyr Glu Leu Phe Glu Lys Asp Gly Asn Arg Pro IleMet Ile Glu Glu 515 520 525 Leu Ala Ser Glu Leu Gly Leu Gly Pro Ser ValPro Val His Val Val 530 535 540 Leu Gln Asp Trp Ile Arg His Ser Asp GlyLys Leu Ser Phe Leu Gly 545 550 555 560 Phe Val Arg Leu Leu His Gly ValSer Ser Arg Thr Leu Gln Lys Ala 565 570 575 20 405 PRT Arabidopsisthaliana 20 Met Ala Ser Val Gly Ile Ala Pro Asn Pro Gly Ala Arg Asp SerThr 1 5 10 15 Gly Val Asp Lys Leu Pro Glu Glu Met Asn Asp Met Lys IleArg Asp 20 25 30 Asp Lys Glu Met Glu Ala Thr Val Val Asp Gly Asn Gly ThrGlu Thr 35 40 45 Gly His Ile Ile Val Thr Thr Ile Gly Gly Arg Asn Gly GlnPro Lys 50 55 60 Gln Thr Ile Ser Tyr Met Ala Glu Arg Val Val Gly His GlySer Phe 65 70 75 80 Gly Val Val Phe Gln Ala Lys Cys Leu Glu Thr Gly GluThr Val Ala 85 90 95 Ile Lys Lys Val Leu Gln Asp Arg Arg Tyr Lys Asn ArgGlu Leu Gln 100 105 110 Thr Met Arg Leu Leu Asp His Pro Asn Val Val SerLeu Lys His Cys 115 120 125 Phe Phe Ser Thr Thr Glu Lys Asp Glu Leu TyrLeu Asn Leu Val Leu 130 135 140 Glu Tyr Val Pro Glu Thr Val His Arg ValIle Lys His Tyr Asn Lys 145 150 155 160 Leu Asn Gln Arg Met Pro Leu IleTyr Val Lys Leu Tyr Thr Tyr Gln 165 170 175 Ile Phe Arg Ala Leu Ser TyrIle His Arg Cys Ile Gly Val Cys His 180 185 190 Arg Asp Ile Lys Pro GlnAsn Leu Leu Val Asn Pro His Thr His Gln 195 200 205 Val Lys Leu Cys AspPhe Gly Ser Ala Lys Val Leu Val Lys Gly Glu 210 215 220 Pro Asn Ile SerTyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu 225 230 235 240 Ile PheGly Ala Thr Glu Tyr Thr Thr Ala Ile Asp Val Trp Ser Ala 245 250 255 GlyCys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro Gly 260 265 270Glu Ser Gly Val Asp Gln Leu Val His Ile Ile Lys Val Leu Gly Thr 275 280285 Pro Thr Arg Glu Glu Ile Lys Cys Met Asn Pro Asn Tyr Thr Glu Phe 290295 300 Lys Phe Pro Gln Ile Lys Ala His Pro Trp His Lys Ile Phe His Lys305 310 315 320 Arg Met Pro Pro Glu Ala Val Asp Leu Val Ser Arg Leu LeuGln Tyr 325 330 335 Ser Pro Asn Leu Arg Ser Ala Ala Leu Asp Thr Leu ValHis Pro Phe 340 345 350 Phe Asp Glu Leu Arg Asp Pro Asn Ala Arg Leu ProAsn Gly Arg Phe 355 360 365 Leu Pro Pro Ala Phe His Phe Lys Pro His GluLeu Lys Gly Val Pro 370 375 380 Leu Glu Met Val Ala Lys Leu Val Pro GluHis Ala Arg Lys Gln Cys 385 390 395 400 Pro Trp Leu Gly Leu 405 21 412PRT Medicago sativa 21 Met Met Ala Ser Gly Gly Val Ala Pro Ala Ser GlyPhe Ile Asp Lys 1 5 10 15 Asn Ala Ser Ser Val Gly Val Glu Lys Leu ProGlu Glu Met Asn Asp 20 25 30 Met Lys Ile Arg Asp Asp Lys Glu Met Glu AlaAla Thr Ile Val Asp 35 40 45 Gly Asn Gly Thr Glu Thr Gly His Ile Ile ValThr Thr Ile Gly Gly 50 55 60 Lys Asn Gly Gln Pro Lys Gln Thr Ile Ser TyrMet Ala Glu Arg Val 65 70 75 80 Val Gly His Gly Ser Phe Gly Val Val PheGln Ala Lys Cys Leu Glu 85 90 95 Thr Gly Glu Thr Val Ala Ile Lys Lys ValLeu Gln Asp Lys Arg Tyr 100 105 110 Lys Asn Arg Glu Leu Gln Thr Met ArgLeu Leu Asp His Pro Asn Val 115 120 125 Val Ser Leu Lys His Cys Phe PheSer Thr Thr Glu Lys Asp Glu Leu 130 135 140 Tyr Leu Asn Leu Val Leu GluTyr Val Pro Glu Thr Val Ser Arg Val 145 150 155 160 Ile Arg His Tyr AsnLys Met Asn Gln Arg Met Pro Met Ile Tyr Val 165 170 175 Lys Leu Tyr SerTyr Gln Ile Cys Arg Ala Leu Ala Tyr Ile His Asn 180 185 190 Ser Ile GlyVal Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val 195 200 205 Asn ProHis Thr His Gln Leu Lys Ile Cys Asp Phe Gly Ser Ala Lys 210 215 220 ValLeu Val Lys Gly Glu Pro Asn Ile Ser Tyr Ile Cys Ser Arg Tyr 225 230 235240 Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu Tyr Thr Thr Ala 245250 255 Ile Asp Ile Trp Ser Ala Gly Cys Val Leu Gly Glu Leu Leu Leu Gly260 265 270 Gln Pro Leu Phe Pro Gly Glu Ser Gly Val Asp Gln Leu Val GluIle 275 280 285 Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Glu Ile Lys CysMet Asn 290 295 300 Pro Asn Tyr Thr Glu Phe Lys Phe Pro Gln Ile Lys AlaHis Pro Trp 305 310 315 320 His Lys Ile Phe His Lys Arg Met Pro Pro GluAla Val Asp Leu Val 325 330 335 Ser Arg Leu Leu Gln Tyr Ser Pro Asn LeuArg Ser Thr Ala Leu Glu 340 345 350 Ala Leu Val His Pro Phe Tyr Asp AspVal Arg Asp Pro Asn Thr Arg 355 360 365 Leu Pro Asn Gly Arg Phe Leu ProPro Leu Phe Asn Phe Lys Val Asn 370 375 380 Glu Leu Lys Gly Val Pro AlaGlu Met Leu Val Lys Leu Val Pro Pro 385 390 395 400 His Ala Arg Lys GlnCys Ala Leu Phe Gly Ser Ser 405 410 22 411 PRT Medicago sativa 22 MetAla Ser Val Gly Val Ala Pro Thr Ser Gly Phe Arg Glu Val Leu 1 5 10 15Gly Asp Gly Glu Ile Gly Val Asp Asp Ile Leu Pro Glu Glu Met Ser 20 25 30Asp Met Lys Ile Arg Asp Asp Arg Glu Met Glu Ala Thr Val Val Asp 35 40 45Gly Asn Gly Thr Glu Thr Gly His Ile Ile Val Thr Thr Ile Gly Gly 50 55 60Arg Asn Gly Gln Pro Lys Gln Thr Ile Ser Tyr Met Ala Glu Arg Val 65 70 7580 Val Gly His Gly Ser Phe Gly Val Val Phe Gln Ala Lys Cys Leu Glu 85 9095 Thr Gly Glu Thr Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Tyr 100105 110 Lys Asn Arg Glu Leu Gln Thr Met Arg Leu Leu Asp His Pro Asn Val115 120 125 Val Ser Leu Lys His Cys Phe Phe Ser Thr Thr Glu Lys Asp GluLeu 130 135 140 Tyr Leu Asn Leu Val Leu Glu Tyr Val Pro Glu Thr Val HisArg Val 145 150 155 160 Ile Lys His Tyr Ser Lys Leu Asn Gln Arg Met ProMet Ile Tyr Val 165 170 175 Lys Leu Tyr Thr Tyr Gln Ile Phe Arg Ala LeuSer Tyr Ile His Arg 180 185 190 Cys Ile Gly Val Cys His Arg Asp Ile LysPro Gln Asn Leu Leu Val 195 200 205 Asn Pro His Thr His Gln Val Lys LeuCys Asp Phe Gly Ser Ala Lys 210 215 220 Val Leu Val Lys Gly Glu Pro AsnIle Ser Tyr Ile Cys Ser Arg Tyr 225 230 235 240 Tyr Arg Ala Pro Glu LeuIle Phe Gly Ala Thr Glu Tyr Thr Thr Ala 245 250 255 Ile Asp Val Trp SerVal Gly Cys Val Leu Ala Glu Leu Leu Leu Gly 260 265 270 Gln Pro Leu PhePro Gly Glu Arg Gly Val Asp Gln Leu Val Glu Ile 275 280 285 Ile Lys ValLeu Gly Thr Pro Thr Arg Glu Glu Ile Lys Cys Met Asn 290 295 300 Pro AsnTyr Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp 305 310 315 320His Lys Ile Phe His Lys Arg Met Pro Ala Glu Ala Val Asp Leu Val 325 330335 Ser Arg Leu Leu Gln Tyr Ser Pro Asn Leu Arg Cys Gln Ala Leu Asp 340345 350 Cys Leu Thr His Pro Phe Phe Asp Glu Leu Arg Asp Pro Asn Ala Arg355 360 365 Leu Pro Thr Gly Arg Phe Leu Pro Pro Leu Phe Asn Phe Lys ProHis 370 375 380 Glu Leu Lys Gly Val Pro Val Glu Thr Leu Met Lys Leu ValPro Glu 385 390 395 400 His Ala Arg Lys Gln Cys Pro Phe Leu Gly Leu 405410 23 407 PRT Arabidopsis thaliana 23 Met Ala Ser Leu Pro Leu Gly ProGln Pro His Ala Leu Ala Pro Pro 1 5 10 15 Leu Gln Leu His Asp Gly AspAla Leu Lys Arg Arg Pro Glu Leu Asp 20 25 30 Ser Asp Lys Glu Met Ser AlaAla Val Ile Glu Gly Asn Asp Ala Val 35 40 45 Thr Gly His Ile Ile Ser ThrThr Ile Gly Gly Lys Asn Gly Glu Pro 50 55 60 Lys Gln Thr Ile Ser Tyr MetAla Glu Arg Val Val Gly Thr Gly Ser 65 70 75 80 Phe Gly Ile Val Phe GlnAla Lys Cys Leu Glu Thr Gly Glu Ser Val 85 90 95 Ala Ile Lys Lys Val LeuGln Asp Arg Arg Tyr Lys Asn Arg Glu Leu 100 105 110 Gln Leu Met Arg ProMet Asp His Pro Asn Val Ile Ser Leu Lys His 115 120 125 Cys Phe Phe SerThr Thr Ser Arg Asp Glu Leu Phe Leu Asn Leu Val 130 135 140 Met Glu TyrVal Pro Glu Thr Leu Tyr Arg Val Leu Arg His Tyr Thr 145 150 155 160 SerSer Asn Gln Arg Met Pro Ile Phe Tyr Val Lys Leu Tyr Thr Tyr 165 170 175Gln Ile Phe Arg Gly Leu Ala Tyr Ile His Thr Val Pro Gly Val Cys 180 185190 His Arg Asp Val Lys Pro Gln Asn Leu Leu Val Asp Pro Leu Thr His 195200 205 Gln Val Lys Leu Cys Asp Phe Gly Ser Ala Lys Val Leu Val Lys Gly210 215 220 Glu Pro Asn Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala ProGlu 225 230 235 240 Leu Ile Phe Gly Ala Thr Glu Tyr Thr Ala Ser Ile AspIle Trp Ser 245 250 255 Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly GlnPro Leu Phe Pro 260 265 270 Gly Glu Asn Ser Val Asp Gln Leu Val Glu IleIle Lys Val Leu Gly 275 280 285 Thr Pro Thr Arg Glu Glu Ile Arg Cys MetAsn Pro Asn Tyr Thr Asp 290 295 300 Phe Arg Phe Pro Gln Ile Lys Ala HisPro Trp His Lys Val Phe His 305 310 315 320 Lys Arg Met Pro Pro Glu AlaIle Asp Leu Ala Ser Arg Leu Leu Gln 325 330 335 Tyr Ser Pro Ser Leu ArgCys Thr Ala Leu Glu Ala Cys Ala His Pro 340 345 350 Phe Phe Asn Glu LeuArg Glu Pro Asn Ala Arg Leu Pro Asn Gly Arg 355 360 365 Pro Leu Pro ProLeu Phe Asn Phe Lys Gln Glu Leu Gly Gly Ala Ser 370 375 380 Met Glu LeuIle Asn Arg Leu Ile Pro Glu His Val Arg Arg Gln Met 385 390 395 400 SerThr Gly Leu Gln Asn Ser 405

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having glycogen synthasekinase activity, wherein the polypeptide comprises at least 400 aminoacids, and wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:10, SEQ ID NO:14, of SEQ ID NO:16 haveat least 90% identity based on the Clustal alignment method with thedefault parameters, or (b) the complement of the nucleotide sequence. 2.The polynucleotide of claim 1, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:10, SEQ ID NO:14,or SEQ ID NO:16 have at least 95% identity based on the clustalalignment method with the default parameters.
 3. The polynucleotide ofclaim 1 comprising the nucleotide sequence of SEQ ID NO:9, SEQ ID NO:13,or SEQ ID NO:15.
 4. The polynucleotide of claim 1, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:10, SEQ IDNO:14, or SEQ ID NO:16.
 5. A chimeric gene comprising the polynucleotideof claim 1 operably linked to a regulatory sequence.
 6. An expressionvector comprising the polynucleotide of claim
 1. 7. A method fortransforming a cell comprising transforming a cell with thepolynucleotide of claim
 1. 8. A cell comprising the chimeric gene ofclaim
 5. 9. A method for producing a plant comprising transforming aplant cell with the polynucleotide of claim 1 and regenerating a plantfrom the transformed plant cell.
 10. A plant comprising the chimericgene of claim
 5. 11. A seed comprising the chimeric gene of claim 5.